Exosomal circRHCG promotes breast cancer metastasis via facilitating M2 polarization through TFEB ubiquitination and degradation

Triple-negative breast cancer (TNBC) is a highly aggressive cancer with distant metastasis. Accumulated evidence has demonstrated that exosomes are involved in TNBC metastasis. Elucidating the mechanism underlying TNBC metastasis has important clinical significance. In the present study, exosomes were isolated from clinical specimens and TNBC cell lines. Colony formation, EdU incorporation, wound healing, and transwell assays were performed to examine TNBC cell proliferation, migration, and metastasis. Macrophage polarization was evaluated by flow cytometry and RT-qPCR analysis of polarization markers. A mouse model of subcutaneous tumor was established for assessment of tumor growth and metastasis. RNA pull-down, RIP and Co-IP assays were used for analyzing molecular interactions. Here, we proved that high abundance of circRHCG was observed in exosomes derived from TNBC patients, and increased exosomal circRHCG indicated poor prognosis. Silencing of circRHCG suppressed TNBC cell proliferation, migration, and metastasis. TNBC cell-derived exosomes promoted M2 polarization via delivering circRHCG. Exosomal circRHCG stabilized BTRC mRNA via binding FUS and naturally enhanced BTRC expression, thus promoting the ubiquitination and degradation of TFEB in THP-1 cells. In addition, knockdown of BTRC or overexpression of TFEB counteracted exosomal circRHCG-mediated facilitation of M2 polarization. Furthermore, exosomal circRHCG promoted TNBC cell proliferation and metastasis by facilitating M2 polarization. Knockdown of circRHCG reduced tumor growth, metastasis, and M2 polarization through the BTRC/TFEB axis in vivo. In summary, exosomal circRHCG promotes M2 polarization by stabilizing BTRC and promoting TFEB degradation, thereby accelerating TNBC metastasis and growth. Our study provides promising therapeutic strategies against TNBC.


INTRODUCTION
Triple-negative breast cancer (TNBC) is characterized by negative expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 1 , accounting for roughly 15% of all cases 2 .TNBC is the most aggressive breast cancer that grows and spreads fast, causing increased risk of recurrence, high metastasis, and a bad outcome 3 .Due to the triple-negative phenotype, the treatment for TNBC is limited, and patients cannot benefit from therapies against these receptors.Even so, the prognosis has been greatly improved in recent years thanks to advances in treatment including surgery, chemotherapy, radiation therapy, and immunotherapy 4,5 .However, exploring the mechanism underlying TNBC metastasis is crucial for understanding TNBC pathogenesis and developing promising therapeutic strategies.
Exosomes are nanosized cell-derived vesicles of ~30-200 nm in diameter that contain abundant cellular constituents such as nucleic acids, serving key roles in intercellular communication and regulating the behavior and activity of recipient cells 6,7 .Intriguingly, growing evidence has shown that exosomes are important regulators of TNBC.A recent study reported that elevated exosomal annexin A2 in the serum of TNBC patients might enhance angiogenesis 8 .Li et al. found that exosomal cluster of differentiation 151 (CD151) promoted TNBC migration and invasion 9 .Additionally, exosomes are implicated in the regulation of drug resistance in TNBC 10 .Thus, exosomes are becoming a promising endogenous vehicle for drug delivery to improve antitumor activities in TNBC.For example, an in vitro study showed that exosomes released by mesenchymal stem cells suppressed aggressiveness in TNBC via delivering microRNA-381-3p (miR-381-3p) 11 .Tumor-released exosomes are key regulators in macrophage polarization.However, exosome-mediated crosstalk between tumor-associated macrophages and tumor cells remains poorly understood.
Circular RNAs (circRNAs) regulate target gene expression and are emerging as vital regulators in cancers.CircRNAs regulate the polarization of tumor-associated macrophages and serve significant roles in tumor metastasis and progression 12 .Although several circRNAs have been reported to act as oncogenes to promote TNBC progression 13,14 , the roles of exosomal circRNA in TNBC need better understanding.Specially, circRNAs are enriched in exosomes and exosomal circRNAs modulate the growth, invasion, and metastasis in cancers including in TNBC 15 .Yang and colleagues found that exosomal circPSMA1 promoted the tumorigenesis and metastasis in TNBC 16 .CircRHCG, termed as hsa_circ_ 0104852, is highly abundant in exosomes derived from TNBC cell line MDA-MB-231 17 .However, the role of exosomal circRHCG in TNBC is still unknown.
Transcription Factor EB (TFEB) is a key transcription factor that controls lysosomal biogenesis and autophagy 18 .Importantly, increased TFEB expression is connected to the aggressiveness of breast cancer 19 .Conversely, knockout of TFEB in macrophages enhanced breast cancer growth via promoting M2 polarization 20 .Thus, elucidating the roles of TFEB in tumor cells and tumorassociated macrophages is important.As exosomes play important roles in shaping tumor microenvironment, we hypothesized that TNBC cell-derived exosomes might regulate macrophage polarization via delivering circRHCG and adjusting TFEB expression, thus regulating TNBC progression.
In summary, we sought to explore the activity of exosomal circRHCG in regulating macrophage and TNBC cell metastasis.In this study, we demonstrated that exosomal circRHCG facilitated M2 polarization by enhancing β-transducin repeat containing E3 ubiquitin protein ligase (BTRC)-dependent ubiquitination and degradation of TFEB, thus promoting cancer cell proliferation, migration, invasion, and metastasis in TNBC.Our study provides a mechanistic insight into exosome-mediated regulation of tumor microenvironment and metastasis in TNBC and suggests potential therapeutic targets and strategies for TNBC.

Increased exosomal circRHCG was associated with poor prognosis in TNBC patients
Exosomes were isolated from blood samples of TNBC patients and volunteers, normal human mammary epithelial MCF10A cells and TNBC cells including MDA-MB-231, BT-549, MDA-MB-468, SUM-159, and MDA-MB-453.Typical vehicles of 30-150 nm in diameter were confirmed by TEM and NTA (Fig. 1a, b), respectively.High expression of exosome markers including CD9, CD63, TSG101, and HSP70 was observed in exosomes, but not in TNBC cells (Fig. 1c).Negative exosome markers Calnexin and GM130 was abundant in TNBC cells, but not in exosomes (Fig. 1c).Ponceau staining was used to confirm that equal amount of proteins from exosomes and TNBC cells were loaded (Supplementary Fig. 1).These observations validated that exosomes were successfully isolated.TNBC patient-derived exosomes showed high levels of circRHCG compared to exosomes from volunteers (Fig. 1d).Besides, TNBC patients with high exosomal circRHCG levels exhibited poor survival (Fig. 1e).Compared to non-tumorigenic epithelial MCF-10A cells, non-TNBC cell lines MCF-7, T47D and BT474 showed moderately increased circRHCG expression, and no significant change was observed in MDA-MB-415 and Skbr-3 cells and exosomes from these non-TNBC cell lines (Fig. 1f, g).However, circRHCG was highly upregulated in TNBC cell lines including MDA-MB-231, BT-549, MDA-MB-468, SUM-159 and MDA-MB-453 and exosomes from these TNBC cell lines (Fig. 1f, g).MDA-MB-468 and MDA-MB-231 cells were chosen for subsequent assays as they showed highest levels of circRHCG.As shown in Fig. 1h, circRHCG (hsa_circ_0104852) is generated from exon 6 to 9 of RHCG gene via back-splicing with a length of 474 bp, and the back-splicing site was verified by Sanger sequencing.As expected, compared to linear RHCG mRNA, circRHCG was highly resistant to RNase R treatment and showed longer half-time after actinomycin D treatment, demonstrating high stability of the circular structure of circRHCG (Fig. 1i, j).Furthermore, we observed that circRHCG was mainly enriched in the cytoplasm, which was validated by fluorescence in situ hybridization (FISH) assays (Fig. 1k, l).These observations suggested high abundance of exosomal circRHCG in TNBC cells and patients.

Knockdown of circRHCG restrained TNBC cell proliferation, migration, invasion and EMT
CircRHCG was knocked down through shcircRHCG transfection, which was confirmed by RT-qPCR (Fig. 2a).Obviously, the colony forming capacity of MDA-MB-468 and MDA-MB-231 cells was impaired by silencing of circRHCG (Fig. 2b).Low EdU incorporation in circRHCG-knockdown cells suggested that knockdown of circRHCG reduced cell proliferation (Fig. 2c).Moreover, silencing of circRHCG significantly restrained TNBC cell migration and invasion (Fig. 2d, e).Furthermore, we found increased E-cadherin expression but decreased expression of N-cadherin, Vimentin, and Snail in circRHCG-silencing cells, suggesting suppression of EMT in these cells (Fig. 2f).These findings indicated that circRHCG might promote TNBC growth and metastasis.

Exosomal circRHCG enhanced the ubiquitination and degradation of TFEB via upregulating BTRC
As TFEB is a vital regulator of breast cancer-associated macrophages 20 , we analyzed TFEB expression in THP-1 cells.The mRNA level of TFEB was unaffected, whereas MDA-MB-468 and MDA-MB-231 -derived exosomes reduced the protein level of TFEB, and exosomal knockdown of circRHCG abrogated the suppressive effect (Fig. 4a, b).Moreover, MG132 treatment also reversed exosome-mediated suppression of TFEB at protein level, indicating that exosomal circRHCG might regulate TFEB expression through the ubiquitin-proteasome mechanism (Fig. 4c).Furthermore, we found that MDA-MB-468 and MDA-MB-231 -derived exosomes markedly enhanced the ubiquitination level of TFEB (Fig. 4d).Coimmunoprecipitation assays showed that BTRC could be immunoprecipitated by an anti-TFEB antibody, suggesting the direct interaction between BTRC and TFEB (Fig. 4e).We determined the levels of ubiquitinated-TFEB and its protein expression after BTRC knockdown in THP-1 cells.Knockdown of BTRC significantly reduced the ubiquitination of TFEB and enhanced TFEB protein expression in THP-1 cells (Fig. 4f, g).MDA-MB-468 and MDA-MB-231-derived exosomes promoted BTRC expression in THP-1 cells, whereas circrRHCG-silencing exosomes did not have this effect (Fig. 4h, i).Our data demonstrated that exosomal circRHCG promoted BTRC expression and subsequently enhanced the ubiquitination and degradation of TFEB in THP-1 cells.

CircRHCG stabilized BTRC mRNA via binding FUS
We predicted that circRHCG contained a potential binding site for FUS, an RNA-binding protein, through RBPDB (http:// rbpdb.ccbr.utoronto.ca)(Fig. 5a).Indeed, FUS could be pulled down by the circRHCG probe, and circRHCG could be enriched by an anti-FUS antibody (Fig. 5b, c).In addition, FISH and immunofluorescence (IF) staining showed colocalization of FUS and circRHCG (Fig. 5d).These data suggested a direct interaction between circRHCG and FUS.Additionally, we found that BTRC mRNA could also be enriched by the anti-FUS antibody, and MDA-MB-468 and MDA-MB-231-derived exosomes further enhanced this enrichment (Fig. 5e, f).As FUS regulates the stability of target mRNAs 21,22 , we analyzed BTRC mRNA stability in response to actinomycin D treatment.MDA-MB-468 and MDA-MB-231-derived exosomes distinctly enhanced the stability of BTRC mRNA (Fig. 5g).These data suggested that exosomal circRHCG might stabilize    BTRC mRNA via binding FUS and facilitating interaction between FUS and BTRC mRNA, thus naturally increasing BTRC expression.
Exosomal circRHCG promoted TNBC cell proliferation and metastasis by facilitating M2 polarization THP-1 cells were stimulated with PMA, treated with TNBC cellderived normal or circRHCG-knockdown exosomes and co-cultured with MDA-MB-231 cells (Fig. 7a).THP-1 cells treated with normal exosomes significantly enhanced colony formation, proliferation, migration and invasion of MDA-MB-231 cells, but THP-1 cells treated with circRHCG-knockdown exosomes did not show these effects (Fig. 7b-e).In addition, normal exosome-treated THP-1 cells    knockdown of circRHCG (Fig. 8b, c).Tumors formed by circRHCHknockdown TNBC cells showed decreased expression of circRHCG and BTRC (Fig. 8d, e).BTRC was downregulated but TFEB was upregulated in tumors by knockdown of circRHCG (Fig. 8f).In addition, knockdown of circRHCG enhanced E-cadherin expression and suppressed the expression of N-cadherin, Vimentin and Snail in tumors (Fig. 8g).Besides, knockdown of circRHCG inhibited the expression of Ki-67, CD163 and Snail (Fig. 8h).To further evaluate circRHCG-silencing-mediated suppression of tumor growth and metastasis, in vivo xenograft mouse models were established by orthotopic mammary fat pad and tail vein injection of circRHCGknockdown MDA-MB-468 and MDA-MB-231 cells.Clodronate liposome was used to deplete macrophages in vivo via intraperitoneal injection.Knockdown of circRHCG or macrophage depletion markedly reduced tumor size, weight, and volume, and circRHCG silencing-mediated anti-tumor activity was further strengthened by macrophage depletion via clodronate liposome administration (Fig. 9a-c).In addition, pulmonary metastasis was reduced by circRHCG knockdown or macrophage depletion, and macrophage depletion further suppressed the metastasis in tumors formed by circRHCG-silencing cells (Fig. 9d).Collectively, these observations suggested that circRHCG contributed to tumor progression and M2 polarization through the BTRC/TFEB axis in vivo.

DISCUSSION
Approximately 2,000,000 new cases of breast cancer a year is reported, and TNBC accounts for about 15% 23,24 .TNBC more commonly affects women aged under 40 and shows lower 5-year survival rate (77%) than other types (93%) 25,26 .Besides survival rate, typically, TNBC patients have bad prognosis, early recurrence, and high risk of distant metastasis that causes great difficulty for treatment 27 .Thus, exploring the mechanisms underlying the metastasis of TNBC is vital for seeking therapies for TNBC.In this study, we found high abundance of circRHCG in TNBC-derived exosomes, and exosomal circRHCG promoted M2 polarization via enhancing BTRC-dependent ubiquitination and degradation of TFEB, thus facilitating tumor cell metastasis and accelerating TNBC progression (Fig. 10).Collectively, circRHCG accelerated TNBC progression via endogenously regulating the behaviors of TNBC cells and exosomal delivery to shape tumor microenvironment.
Exosomes are key regulators in intercellular communication via delivering cellular components.Exosomes secreted by cancer cells can be internalized by various cells, such as tumor cells 28 , fibroblasts 29 , and leukocytes 30 , which modulates the activity of recipient cells and shapes the tumor microenvironment that regulates tumor progression.In particular, accumulated evidence has shown that tumor cell-derived exosomes can educate macrophages into M2 tumor-associated macrophages, accelerating angiogenesis and metastasis 31 .Piao et al. reported that TNBCderived exosomes induced M2 polarization and thus promoted tumor growth and metastasis 32 .Consistently, we found that exosomes released by TNBC cells facilitated M2 polarization and enhanced TNBC cell proliferation and metastasis.Owing to low immunogenicity and great biocompatibility, exosomes show great potential as a drug delivery system for tumor intervention 33 .Extracellular vesicles from TNBC cells carrying CCL5 on their surface influenced the behaviors of resident macrophages, rendering them pro-metastatic in nature, which ultimately led to tumorigenesis 34 .However, a recent study reported that TNBCderived CSF-1-bearing extracellular vesicles promoted proinflammatory macrophages that was correlated with better clinical outcome 35 .These contrary results may attribute to various tumor microenvironment, but it needs further investigation.
CircRNAs are emerging as important regulators in cancers, including TNBC, exerting antitumor or oncogenic activities 36,37 .The abundance of circRNAs and their biological activities in carcinogenesis have been confirmed 38 .Li and colleagues found that exosomal circPDE8A released by tumor cells enhanced invasive growth in pancreatic cancer 39 .However, the roles of exosomal circRNAs in TNBC are largely unknown.In a previous study, researchers demonstrated the abundance of circRHCG (hsa_circ_ 0104852) in exosomes from MDA-MB-231 cells and breast cancer patients 17 , but its roles in TNBC has not been investigated.Here, we validated the presence of circRHCG in TNBC cell and patient-derived exosomes and found the association between exosomal circRHCG and TNBC patient survival.Furthermore, we demonstrated that TNBC cell-derived exosomal circRHCG promoted M2 polarization to enhance TNBC cell growth and metastasis, identifying an oncogenic activity of exosomal circRHCG in TNBC.
CircRNAs exert functions through various mechanisms including sponging miRNAs, interacting with RNA binding proteins and modulating gene transcription 40 .Specially, we found a binding site for the RNA-binding protein FUS and confirmed the interaction between circRHCG and FUS.FUS is a key regulator in maintaining RNA stability.FUS maintains GluA1 mRNA stability by binding it and controlling the length of poly (A) tail 21 .Consistently, we found that FUS interacted with BTRC mRNA and enhanced the stability of BTRC mRNA, naturally upregulating BTRC.
TFEB is an important regulator of macrophage polarization in the tumor microenvironment.Macrophages with reduced TFEB displaying M2 macrophage characteristics significantly accelerated tumor growth and angiogenesis 41 .Herein, we observed reduced expression of TFEB in macrophages after TNBC cellderived exosome treatment, and overexpression of TFEB inhibited M2 polarization, suggesting that exosomal circRHCG-mediated regulation of M2 polarization was dependent on TFEB.However, the mechanism by which knockdown of TFEB enhanced M2 polarization in TNBC is unknown, which needs further investigation.During exploring the mechanism underlying exosomal circRHCG-mediated reduction of TFEB in macrophages, we found that TFEB were reduced at protein levels, but not mRNA levels, indicating circRHCG-mediated posttranslational modification of TFEB.Furthermore, increased BTRC expression was observed.It has been demonstrated that BTRC is pivotal for ubiquitination and degradation of various targets, such as phosphorylated inhibitor of NF-κB α isoform (IκBα), GLI family zinc finger 2 (Gli2) and β-catenin [42][43][44] .In consistence, we demonstrated that BTRC interacted with TFEB to promote its ubiquitination and degradation.
Taken together, we demonstrated that exosomal circRHCG from TNBC cells promoted TNBC progression via facilitating M2 polarization through BTRC-dependent ubiquitination and degradation of TFEB.Exosome-derived circRHCG accelerated TNBC invasion and metastasis, implying its potential as a therapeutic target for TNBC treatment and diagnostic and prognostic biomarker.

Clinical specimens
Blood samples were collected from forty patients diagnosed with TNBC and forty healthy volunteers at the First Affiliated Hospital of Nanjing Medical University used for exosome isolation.Patients and volunteers provided written consent.All clinical studies complied with relevant ethical regulations including the Declaration of Helsinki and were approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (Ethics code 2022-SR-520).

Isolation and characterization of exosomes
Whole blood was collected and allowed to clot, and serum was collected after centrifugation for exosome extraction with Total Exosome Isolation Reagent (from serum, Thermo Fisher Scientific, Waltham, MA, USA).TNBC cells (3 × 10 6 ) were seeded and maintained for 24 hours, and the medium was replaced with fresh exosome-depleted medium.After 48 hours, the medium was harvested for exosome isolation using Total Exosome Isolation Reagent (from cell culture media, Thermo Fisher Scientific).The morphology, size, and concentration of exosomes were examined by transmission electron microscopy (TEM) and NanoSight NS300 (Malvern, UK), respectively.The expression of exosome markers including CD63, CD9, tumor susceptibility gene 101 (TSG101) and heat shock protein 70 (HSP70) and negative markers including Calnexin and GM130 was examined by Western blotting.

Internalization of exosomes
Exosomes were stained with PKH-26 (2 µM, Sigma, St. Louis, MO, USA) for 30 min and washed twice in PBS to remove excessive PKH-26 dye.Subsequently, THP-1 cells were co-cultured with PKH-26-labeled exosomes or an equal volume of the PKH26-PBS control for 24 h and stained with DAPI (1 µg/mL, Beyotime, Shanghai, China) prior to examination under a fluorescence microscope.For co-staining of circRHCG and exosomes, Cy3labeled circRHCG was transfected into MDA-MB-468 and MDA-MB-231, and exosomes were extracted from culture supernatants and labeled with PKH67 (Sigma) at 2 µM for 30 min.THP-1 cells were incubated with exosomes for 24 h and stained with DAPI (1 µg/mL, Beyotime) prior to examination under a fluorescence microscope.

Characterization of circRHCG
RNA was isolated and treated with RNase R (5 U/μg, Abcam, Cambridge, UK) or mock for 1 h at 37 °C.The abundance of Rh family C glycoprotein (RHCG) mRNA and circRHCG was detected with quantitative reverse transcription PCR (RT-qPCR).For inhibition of gene transcription, cells were treated with actinomycin D (Selleck) at 2 μg/mL for 0, 4, 8, 12, 16, and 20 h prior to RNA isolation, and RHCG mRNA and circRHCG was examined by RT-qPCR.Divergent primers were designed to amplify circRHCG, and the back-splicing site of circRHCG was confirmed by Sanger sequencing.
Colony formation TNBC cells (1 × 10 3 ) were treated and maintained for 14 d in serum-free DMEM in a cell incubator.Cell colonies were fixed and stained with crystal violet (1%, Sigma).Subsequently, cell colonies were rinsed and imaged.

EdU incorporation
EdU incorporation was performed using the Click-IT EdU kit (Thermo Fisher Scientific).Briefly, cells were treated and incubated in fresh culture medium containing 10 µM of EdU for 2 h.Subsequently, cells were fixed, permeabilized and incubated in Click-iT reaction cocktail solution for 30 min.After wash, cells were mounted with antifade mountant with DAPI (Thermo Fisher Scientific).
Wound healing assay TNBC cells were seeded in 6-well plates in completed DMEM medium and cultured to a monolayer with 90% confluence.A wound of 1 mm was scratched using a pipette tip.Subsequently, cells were washed in PBS and maintained in serum-free DMEM medium for 24 h in a cell incubator.Wound healing was evaluated with an Olympus BX51 microscope (Tokyo, Japan).

Transwell assay
Transwell chambers (BD, Franklin Lakes, NJ, USA) were applied for evaluating cell invasion.Matrix gel was pre-coated on the upper chamber.After indicated treatment, cells were seeded and incubated for 24 h.Cells which invaded into the lower chamber were fixed, stained with crystal violet (1%, Sigma) and imaged under an Olympus BX51 microscope.
RNA pull-down assay THP-1 cells were lysed, and cell lysates were collected.Biotinylated circRHCG and random probes (RiboBio) were mixed thoroughly with cell lysates and incubated overnight at 4 °C.Streptavidin magnetic beads (Thermo Fisher Scientific) were added into samples and incubated for 2 h with gentle rotation.Subsequently, protein was recovered from the complexes pulled down by probes and applied for Western blotting analysis of FUS using a rabbit anti-FUS antibody (1:500, ab243880, clone: BLR023E, Abcam).

RNA immunoprecipitation
THP-1 cells were lysed, and cell lysates were collected.An anti-FUS antibody (5 µg, ab243880, clone: BLR023E, Abcam) or normal IgG (5 µg, Abcam) was mixed with cell lysates and incubated overnight.Protein A/G magnetic beads (Sigma) were added into samples and incubated for additional 1 h.Subsequently, RNA was recovered, and the abundance of circRHCG and BTRC mRNA was examined with RT-qPCR.

Fig. 1
Fig. 1 Increased exosomal circRHCG was associated with poor prognosis.a TEM examination of exosomes.b NTA analysis of exosome size.c Western blotting analysis of CD63, CD9, TSG101, HSP70, Calnexin, GM130, GAPDH and β-actin in TNBC cells and exosomes.d RT-qPCR analysis of circRHCG in exosomes derived from volunteers (n = 40) and TNBC patients (n = 40).e Overall survival of patients (circRHCG high, n = 20; circRHCG low, n = 20).f, g RT-qPCR analysis of circRHCG in breast cancer cell lines and exosomes derived from these cell lines (n = 3).h Genomic loci of circRHCG and its back-splicing site.i, j RT-qPCR analysis of RHCG mRNA and circRHCG after RNase R digestion and actinomycin D treatment (n = 3).k RNA levels of GAPDH, U6 and circRHCG in the nuclear and cytosolic fractions (n = 3).l FISH assays for analysis of circRHCG localization with Cy3-conjugated probes (Red).DAPI (Blue) was used for nuclear staining.Scale bar, 25 µm.*p < 0.05, **p < 0.01 and ***p < 0.001.Error bars indicate SD.All results are representative of or combined from at least three independent experiments.

Fig. 4
Fig. 4 Exosomal circRHCG enhanced the ubiquitination and degradation of TFEB via upregulating BTRC.a, b The mRNA and protein levels of TFEB (n = 3).c Western blotting analysis of TFEB in THP-1 cells treated with exosomes (n = 3).MG132 was used to inhibit proteasome.d Ubiquitination analysis of immunoprecipitated TFEB from THP-1 cells treated with exosomes.e BTRC was coimmunoprecipitated with TFEB.f Ubiquitination analysis of immunoprecipitated TFEB from THP-1 cells after BTRC knockdown.g Protein levels of BTRC and TFEB in THP-1 cells (n = 3).h, i The mRNA and protein levels of BTRC (n = 3).***p < 0.001.Error bars indicate SD.All results are representative of or combined from at least three independent experiments.

Fig. 5
Fig. 5 CircRHCG stabilized BTRC mRNA via binding FUS. a A binding site for FUS in circRHCG.b FUS was pulled down by the circRHCG probe, not by the random probe.c CircRHCG was enriched by the anti-FUS antibody (n = 3).d Colocalization of circRHCG (Red) and FUS (Green) was confirmed by combined FISH and IF staining.e BTRC mRNA was enriched by the anti-FUS antibody (n = 3).f The enrichment of BTRC mRNA by the anti-FUS antibody in THP-1 cells treated with exosomes (n = 3).g The mRNA levels of BTRC in THP-1 cells treated with exosomes in response to actinomycin D (n = 3).*p < 0.05, **p < 0.01 and ***p < 0.001.Error bars indicate SD.All results are representative of or combined from at least three independent experiments.

Fig. 9
Fig. 9 Knockdown of circRHCG inhibited tumor growth and metastasis.a-c MDA-MB-468 and MDA-MB-231 cells were orthotopically injected into the mammary fat pad, and tumors were imaged and weighed.Tumor volume was monitored (n = 6 each group).d MDA-MB-468 and MDA-MB-231 cells were injected into nude mice via tail vein.The lung was excised and imaged.Pulmonary metastasis was examined via H&E staining, and metastatic nodules were counted (n = 6 each group).*p < 0.05, **p < 0.01, and ***p < 0.001.Error bars indicate SD.

Fig. 10
Fig. 10 The schematic diagram of exosomal circRHCG-mediated regulation of M2 polarization and tumor progression in TNBC.Exosomal circRHCG binds to FUS and stabilizes BTRC mRNA to promote TFEB ubiquitination and degradation, thereby promoting TNBC progression by facilitating M2 polarization.